The present invention relates generally to the field of diagnostic compositions and methods useful in the diagnosis of Lyme borreliosis.
The bacterium Borrelia burgdorferi (sensu lato) is the causative agent of Lyme borreliosis, i.e., Lyme disease. This disease is transmitted by the bite of various species of Ixodes ticks carrying the spirochete. The main reservoir of the infection in the United States is the white footed mouse, Peromyscus leucopus, and the infection can be transmitted to many mammalian species including dogs, cats, and man [J. G. Donahue, et al., Am. J. Trop. Med. Hyg., 36:92-96 (1987); R. T. Green, et al, J. Clin. Micro., 26:648-653 (1988)]. Despite the presence of an active immune response, the disease persists for years in patients. Such persistence is postulated to be the result, at least in part, of antigenic variation in the bacterial proteins [J. R. Zhang et al, Cell, 89:275-285 (1997)].
Publications relating to proteins and polypeptides of Borrelia burgdorferi having suggested their use as diagnostic or pharmaceutical agents. Such proteins and polypeptides include outer surface proteins A and B (OspA and OspB), flagellin, and other proteins designated P21, P39, P66, and P83 according to their estimated molecular weights [A. G. Barbour et al, Infect. Immun., 45:94-100 (1984); W. J. Simpson et al., J. Clin. Microbiol., 28:1329-1337 (1990); K. Hansen et al., Infect. Immun., 56:2047-2053 (1988); K. Hansen et al., Infect. J. Clin. Microbiol., 26:338-346 (1988); B. Wilske et al., Zentral, Bakteriol, Parsitenkd, Infektionshkr, Hyg. Abt. 1 Orig. Reihe, A., 263:92-102 (1986); D. W. Dorward et al., J. Clin. Microbiol., 29:1162-1170 (1991); published NTIS U.S. patent application No. 485,551; European patent application No. 465,204, published Jan. 8, 1992; International Patent Application No. PCT/US91/01500, published Sep. 19, 1991; International Patent Application No. PCT/EP90/02282, published Jul. 11, 1991; International Patent Application No. PCT/DK89/00248, published May 3, 1990; International patent application No. WO92/00055, published Jan. 9, 1992.]
However, the diagnosis of Lyme disease in humans and animals has been compromised by the lack of definitive serology leading to rapid and accurate testing. Current diagnostic tests suffer from low sensitivity and specificity, as illustrated by a recent survey of diagnostic laboratories"" performance issued by the Wisconsin State Laboratory of Hygiene [L. Bakken et al., J. Clin. Microbiol., 35:537 (1997)].
There is thus a need in the art for a simple, sensitive and specific diagnostic composition and method for early detection of Lyme disease.
The present invention satisfies the need in the art by providing methods and compositions which permit rapid and accurate detection of Lyme disease. The methods and compositions of the invention advantageously avoid serologic cross-reactivity with other conditions, including syphilis, chronic arthritis, and multiple sclerosis, from which differential diagnosis was required using prior art methods.
In one aspect, the invention provides a peptide having the amino acid sequence MKKDDQIAAAMVLRGMAKDGQALKD [SEQ ID NO:1], termed herein IR6, which is an invariable region which is immunodominant in human and animal Lyme disease patients.
In another aspect, the invention provides an artificial protein which contains the amino acid sequence of IR6, or an analog, homolog or fragment thereof. In one embodiment, this protein may be a fusion protein containing IR6 or a fragment thereof and a fusion partner. In one particularly desirable embodiment, the fusion protein contains a fragment of IR6 corresponding to a T cell epitope, wherein the T cell epitope is fused to a one of the other five invariable regions identified herein (IR1-IR5).
In another desirable embodiment, the artificial protein contains the amino acid sequence of IR6 with an N-terminal amino acid suitable for biotinylation.
In yet another aspect, the invention provides a nucleic acid sequence encoding IR6 peptide or a protein of the invention. In still another aspect, the invention provides a vector comprising a nucleic acid sequence according to the invention under the control of suitable regulatory sequences. In a further aspect, the invention provides a host cell transformed with the vector of the invention.
In a still further aspect, the present invention provides a diagnostic reagent comprising a nucleic acid sequence of the invention and a detectable label which is associated with said sequence.
In yet a further aspect, the invention provides an isolated antibody which is specific for the IR6 peptide of the invention or a fragment thereof. In still a further aspect, the invention provides a diagnostic reagent comprising the antibody of the invention.
In yet another aspect, the invention provides an anti-idiotype antibody specific for the anti-IR6 antibody of the invention and a diagnostic reagent containing the anti-idiotype antibody with a detectable label linked thereto.
In yet a further aspect, the invention provides a method for diagnosing Lyme disease in a human or animal. This method includes the steps of incubating an antigen or antibody of this invention, preferably conventionally labeled for detection, with a sample of biological fluids from a human or an animal to be diagnosed. In the presence of B. burgdorferi infection of the human or animal patient, an antigen-antibody complex is formed. Subsequently the reaction mixture is analyzed to determine the presence or absence of these antigen-antibody complexes. In a further embodiment, the diagnostic assay employs DNA sequences, preferably anti-sense sequences, of the antigen or fragments thereof, and diagnoses infection by the presence of sequences in a biological fluid from the patient that hybridizes thereto. Other conventional assay formats may be employed using reagents identified by this invention.
In another aspect the invention provides a kit for diagnosing infection with B. burgdorferi in a human or an animal patient sample which contains at least one antibody capable of binding at least one antigen of this invention of antigenic fragment(s) thereof, or a DNA sequence encoding one or more antigen(s) of this invention or an anti-sense sequence thereof. The antibodies and sequences may be optionally labeled for detection, or a detection system may be included in the kit.
In another aspect, the invention provides a therapeutic composition and methods for treating humans and/or animals with Lyme disease. The therapeutic composition contains an antibody, or protein, or fragment as described above and a suitable pharmaceutical carrier.
In a further aspect, the invention provides vaccine compositions and methods of vaccinating a human or animal patient against Lyme Disease by use of these above-described compositions. The vaccine composition may contain the IR6 protein, fragments thereof, fusion proteins or mixtures of proteins as described above with a pharmaceutically acceptable carrier. More preferably, the vaccine compositions contain fusion proteins composed of IR6, or a fragment thereof corresponding to a T cell epitope, fused to a partner such as IR1-IR5.
In yet a further aspect, the invention provides vaccine compositions and methods of vaccinating a human or animal patient against Lyme Disease by use of nucleic acid compositions, e.g. DNA vaccines. The compositions contain an effective amount of a DNA sequence encoding at least one IR1-5 or IR6 peptide or protein of this invention and a pharmaceutically acceptable carrier.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.